Delft University of Technology

Serpentinimonas gen. Nov., serpentinimonas raichei sp. nov., serpentinimonas barnesii sp. nov. and serpentinimonas maccroryi sp. nov., hyperalkaliphilic and facultative autotrophic isolated from terrestrial serpentinizing springs

Bird, Lina J.; Kuenen, J. Gijs; Osburn, Magdalena R.; Tomioka, Naotaka; Ishii, Shun’Ichi; Barr, Casey; Nealson, Kenneth H.; Suzuki, Shino DOI 10.1099/ijsem.0.004945 Publication date 2021 Document Version Final published version Published in International Journal of Systematic and Evolutionary Microbiology

Citation (APA) Bird, L. J., Kuenen, J. G., Osburn, M. R., Tomioka, N., Ishii, SI., Barr, C., Nealson, K. H., & Suzuki, S. (2021). Serpentinimonas gen. Nov., serpentinimonas raichei sp. nov., serpentinimonas barnesii sp. nov. and serpentinimonas maccroryi sp. nov., hyperalkaliphilic and facultative autotrophic bacteria isolated from terrestrial serpentinizing springs. International Journal of Systematic and Evolutionary Microbiology, 71(8), [004945]. https://doi.org/10.1099/ijsem.0.004945 Important note To cite this publication, please use the final published version (if applicable). Please check the document version above.

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This work is downloaded from Delft University of Technology. For technical reasons the number of authors shown on this cover page is limited to a maximum of 10. TAXONOMIC DESCRIPTION Bird et al., Int. J. Syst. Evol. Microbiol. 2021;71:004945

DOI 10.1099/ijsem.0.004945 OPEN ACCESS

Serpentinimonas gen. nov., Serpentinimonas raichei sp. nov., Serpentinimonas barnesii sp. nov. and Serpentinimonas maccroryi sp. nov., hyperalkaliphilic and facultative autotrophic bacteria isolated from terrestrial serpentinizing springs

Lina J. Bird1,2, J. Gijs Kuenen2,3, Magdalena R. Osburn4, Naotaka Tomioka5, Shun’ichi Ishii5,6, Casey Barr2, Kenneth H. Nealson2 and Shino Suzuki5,6,7,*

Abstract Three highly alkaliphilic bacterial strains designated as A1T, H1T and B1T were isolated from two highly alkaline springs at The Cedars, a terrestrial serpentinizing site. Cells from all strains were motile, Gram-negative­ and rod-shaped.­ Strains A1T, H1T and B1T were mesophilic (optimum, 30 °C), highly alkaliphilic (optimum, pH 11) and facultatively autotrophic. Major cellular fatty acids were saturated and monounsaturated hexadecenoic and octadecanoic acids. The genome size of strains A1T, H1T and B1T was 2 574 013, 2 475 906 and 2 623 236 bp, and the G+C content was 66.0, 66.2 and 66.1 mol%, respectively. Analysis of the 16S rRNA genes showed the highest similarity to the genera (95.1–96.4 %), (93.0–93.6 %) and Hydrogenophaga (93.0–96.6 %) in the family . Phylogenetic analysis based on 16S rRNA gene and phylogenomic analysis based on core gene sequences revealed that the isolated strains diverged from the related species, forming a distinct branch. Average amino acid identity values of strains A1T, H1T and B1T against the genomes of related members in this family were below 67 %, which is below the suggested threshold for genera boundaries. Average nucleotide identity by blast values and digital DNA– DNA hybridization among the three strains were below 92.0 and 46.6 % respectively, which are below the suggested thresh- olds for species boundaries. Based on phylogenetic, genomic and phenotypic characterization, we propose Serpentinimonas gen. nov., Serpentinimonas raichei sp. nov. (type strain A1T=NBRC 111848T=DSM 103917T), Serpentinimonas barnesii sp. nov. (type strain H1T= NBRC 111849T=DSM 103920T) and Serpentinimonas maccroryi sp. nov. (type strain B1T=NBRC 111850T=DSM 103919T) belonging to the family Comamonadaceae. We have designated Serpentinimonas raichei the type species for the genus because it is the dominant species in The Cedars springs.

The family Comamonadaceae, which belongs to the class hydrogen oxidizers [11], and phosphate-­accumulating and , was first described by Willems et al. [1] -removing bacteria [6, 12], and cyclohexane-degrading­ bacte- and now contains at least 50 genera. Most members of this ria [13]. In this study, we characterize three hyper-­alkaliphilic family were isolated from soil, freshwater, activated sludge, strains (A1T, H1T and B1T) isolated from an active terrestrial hot springs and pond water [2–8]. These genera harbour a serpentinization site that represents an unusual microbial phenotypic diversity that includes aerobic organotrophs [2], habitat, as the fluids are highly alkaline, enriched in calcium, anaerobic denitrifiers [5, 9], Fe3+-­reducing bacteria [10], low in sodium and have abundant dissolved hydrogen gas

Author affiliations: 1Center for Bio/Molecular Science and Engineering Naval Research Lab, 4555 Overlook Ave S.W., Washington DC 20375, USA; 2Department of Earth Sciences, University of Southern California, 35 W. 37th St. SHS 560, Los Angeles, California 90089, USA; 3Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629HZ, Delft, Netherlands; 4Department of Earth and Planetary Sciences, Weinberg College of Arts & Sciences. Northwestern University Evanston, Evanston, USA; 5Kochi Institute for Core Sample Research, Japan Agency for Marine-­ Earth Science and Technology (JAMSTEC), Monobe B200, Nankoku, Kochi 783-8502, Japan; 6Institute for Extra-­cutting-­edge Science and Technology Avant-­garde Research (X-star),­ JAMSTEC, Natsushima 2-15, Yokosuka, Kanagawa 237-0061, Japan; 7Institute of Space and Astronautical Science (ISAS), Japan Aerospace Exploration Agency (JAXA), 3-1-1 Yoshinodai, Chuo-ku,­ Sagamihara, Kanagawa 252-5210, Japan. *Correspondence: Shino Suzuki, suzuki.​ shino2@​ jaxa.​ jp​ Keywords: alkaliphile; autotrophic growth; Serpentinization; Serpentinomonas/Serpentinimonas. Abbreviations: AAI, average amino acid identity; ANIb, average nucleotide identity by blast; BS, Barnes Spring; CSM, Cedars standard medium; dDDH, digital DNA–DNA hybridization. The GenBank accession number for the 16S rRNA gene sequence of strain A1T is MT882023, for H1T it is MW414531 and for B1T it is MW411452. The genome sequence accession number of A1T is AP014568.1, for H1T it is BAWN00000000.1 and for B1T it is AP014569.1 for the chromosome and AP014570.1 for a plasmid. 004945 © 2021 The Authors This is an open-­access article distributed under the terms of the Creative Commons Attribution License.

1 Bird et al., Int. J. Syst. Evol. Microbiol. 2021;71:004945

[14–16]. Given that relatives of strains A1T, H1T and B1T neighbour-­joining. Phylogenetic analysis based on 16S rRNA were dominant in various terrestrial active serpentinizing genes revealed that strains A1T, H1T and B1T belong to the sites, these strains are likely relevant to the geochemistry of family Comamonadaceae in the order . The terrestrial serpentinization sites. We expect future studies on three isolates formed a distinct branch separate from other the ecology, physiology, and molecular genetics members of Comamonadaceae with the bootstrap value of of these organisms to contribute to a better understanding of 100 (Fig. 1). The closest relatives of the three isolates were life under these extremely alkaline conditions. the genera Macromonas [4], Malikia [6] and Hydrogenophaga [11]. The samples were collected from Barnes Spring 1 (BS1) and Barnes Spring 5 (BS5; elevation 282 m, N: 38° 37.282′, W: The genome sizes of strains A1T, H1T and B1T were 2 574 013, 123° 07.987′) located at The Cedars serpentinization site in 2 475 906 and 2 623 236 bp, and the G+C contents were 66.0, northern California as described previously [17, 18]. Briefly, 66.2 and 66.1 mol%, respectively. CheckM analysis showed samples of pre-autoclaved­ glass beads (0.11 mm diameter that the genome completeness of the strains was over 99 % ballotini beads) that were incubated in situ in the BS1 pool at [22]. Thirty conserved marker genes were extracted from the

Eh of around −250 mV for 1 week and then collected for the 14 genomes in the family Comamonadaceae and Burkholderia isolation of strains A1T and B1T. Strain H1T was isolated from cepacia in the family Burkholderiaceae, and a concatenated a sample of pool BS5 water. The samples were inoculated in alignment (5823–6689 amino acids) was generated in the sterile Cedars standard medium (CSM) 1 containing 0.05 mm CheckM platform [23]. A maximum-likelihood­ phylog-

Na2SO4, 0.378 mm NH4Cl, 0.05 mm MgCl2, 0.06 mm K2HPO4, enomic tree based on the concatenated alignment was gener- 10 mm CABS or Na2CO3/NaHCO3, 2 mm CaCO3 (as suspen- ated by using mega X [24] and the JTT matrix-based­ model sion), 4 mm sodium acetate, 10 ml l−1 of ATCC trace mineral with 100 resamples. This phylogenomic tree indicated that supplement and 10 ml l−1 of ATCC vitamin solution. The pH strains A1T, H1T and B1T are deeply branching among those was adjusted to pH 10.5–11.2 using NaOH. Gas phase of the of closely related genera, as seen in the tree based on 16S media in stoppered serum vials was replaced with a mixture rRNA genes (Fig. 2). of O /H /N /Ar (2.6 : 50 : 9.8 : 37.6 by volume at 1 atm). After 2 2 2 Amino acid identity (AAI) values of the strains against the incubation for 2 weeks at 16–18 °C, the sample suspension genomes of closely related genera in the family Comamona- was streaked on CSM1 agar plates containing CSM1 with 2 % daceae were obtained using the Kostas lab AAI calculator prewashed noble (Difco) or Korean (Daishin) agar at pH 10.5, web server (http://​enve-​omics.​ce.​gatech.​edu/​aai/) [23]. In the and incubated under the same gas mixture. Streaked plates past, AAI values between 60–80 % were taken as thresholds were incubated at 18 °C for 2 weeks. for distinguishing genera [25, 26]. However, recent studies of Creamy/opaque colonies of strains A1T, B1T and H1T were new genus descriptions in the family Comamonadaceae and purified after repeatedly culturing on the CSM1 agar plates other phylum proposed that the threshold for genera bounda- at 18 °C for 2 weeks. Colonies of strains A1T and B1T were ries should be 70 % for AAI [8, 27]. The three isolated strains suspended in glycerol diluted by CSM1 (20 % v/v) and stored showed the highest AAI values to genus Macromonas, with at −80 °C. Strain H1T purified on the plate was incubated at a value of 67 %. The next highest AAI values were 65–66 % 16 °C in liquid CSM1 and concentrated by centrifugation and with the genus Malikia, while AAI values with the genus then stored in glycerol diluted by CSM1 (20 % v/v) at −80 °C. Hydrogenophaga were 64–65 % (Table 1). All the AAI values were lower than the proposed genus boundary threshold [27], DNA extraction from the strains and determination of making the strains A1T, H1T and B1T distinct from previously genome sequences have been described previously [19]. The described genera. complete genomes of two strains, A1T and B1T, and the draft genome of strain H1T have been reported previously [19]. Average nucleotide identity by blast (ANIb) values and The 16S rRNA gene nucleotide sequences of the strains were digital DNA–DNA hybridization (dDDH) were calculated retrieved from the genome sequences and aligned with 24 by using JSpecies [28] and the DSMZ Genome-­to-­Genome reference sequences from public databases using the program Distance Calculator platform [29], respectively. The ANIb muscle [20]. Strains A1T, H1T and B1T were closely related and dDDH values among the three isolated strains were to the type species of three genera including 85.0–92.0 % and 28.8–46.6 %, respectively (Table 1), which P1T (95.3, 96.4, 96.3 % 16S rRNA gene sequence identities, are lower than the delineation of species boundaries (ANIb respectively), DSM 12705T (93.0, <95 % and dDDH <70 %) [30]. 93.5, 93.6 %) and Hydrogenophaga flava DSM 619T (95.3, 96.3, The purified strains were initially grown and tested on CSM2, 96.6 %). Among the three strains, 16S rRNA gene sequence which contains 0.1 mm Na SO , 0.755 mm NH Cl, 0.1 mm identity between strains A1T and H1T was 98.9 %, between 2 4 4 MgCl , 0.23 mm K HPO , 20 mm CaCO (as suspension), strains A1T and B1T it was 97.6%, and between strains B1T 2 2 4 3 15 mm CAPS buffer (pH 11), 10 ml l−1 ATCC trace mineral and H1T it was 98.7 %. supplement and 10 ml l−1 ATCC vitamin solution. The portion A phylogenetic tree was created by using the maximum-­ for the liquid and gas phase was approximately 35 : 65. The likelihood method with RaxML [21]. The robustness of gas composition was H2/N2/air 35 : 35 : 30, the temperature furcated branches was supported by bootstrap values (1000 for the cultivations is at 30 °C. Further optimization led to replicates) (Fig. 1). The topology was further confirmed by CSM3, which contained 0.2 mm Na2SO4, 1.5 mm NH4Cl,

2 Bird et al., Int. J. Syst. Evol. Microbiol. 2021;71:004945

Fig. 1. Phylogenetic relationship derived from 16S rRNA gene sequences between strains A1T, B1T and H1T and other related taxa of the family Comamonadaceae. The tree was reconstructed using the maximum-likelihood­ method based on 16S rRNA gene sequences. Bootstrap values greater than 80 % are shown at branch points. Bar, 0.02 substitutions per nucleotide position.

−1 T 0.199 mm MgCl2, 0.23 mm K2HPO4, 5 mm CaCl2, 10 ml l strain A1 grew poorly in the absence of hydrogen. None ATCC vitamin solution, 10 ml l−1 ATCC mineral solution and of the strains could utilize sulphate, iron (III) hydroxide, or 15 mm CAPS buffer (pH 11) and the gas composition was iron (II/III) oxide as electron acceptors. All strains grew best

H2/N2/air 35 : 35 : 30 for autotrophic growth and N2/air 75 : 30 aerobically on sub-­atmospheric levels of oxygen (1–4 % v/v). for heterotrophic growth. Calcium, phosphate, vitamins and During mixotrophic growth on hydrogen and acetate, the minerals were added as separate filter-sterilized­ solutions after minimum doubling time was 10 h for strain A1T, 12.5 h for autoclaving the basal salt solution. For autotrophic growth, the strain H1T and 8.5 h for strain B1T, respectively. CaCl in CSM3 was replaced with 20 mm CaCO , while 3 mm 2 3 sensitivity was tested both on solid and in liquid acetate was used for routine heterotrophic growth. Substrate medium for kanamycin at 50 µg ml−1 and for gentamycin utilization were tested in CMS2. pH range and optimum pH at 10 µg ml−1. Growth on plates was assessed visually, while were tested in both CSM2 and CSM3. Growth rate determina- growth in liquid medium was monitored via protein concen- tion, antibiotic sensitivity, catalase assay, Gram staining, light trations using the Lowry assay [31]. All three strains were microscopy, TEM and SEM, cytochrome analysis, and lipid sensitive to both kanamycin and gentamycin, showing no analysis were performed in CSM3. When testing utilization growth on plates or in liquid medium. of organic substrates, a substrate was added to the CSM2 and CSM3. For testing the anaerobic growth, medium was Fatty acid content for strains A1T and B1T were analysed thoroughly flushed with 2N . The results of substrate utilization from acetate grown chemostat cultures. Cells were pelleted are shown in Table 2. All three strains were able to grow auto- by centrifugation and stored at −20 °C. Cells for H1T were trophically using hydrogen, calcium carbonate and oxygen or grown in batch culture on acetate and harvested in the heterotrophically on a variety of electron donors, although same way. Membrane lipids were extracted via a modified

3 Bird et al., Int. J. Syst. Evol. Microbiol. 2021;71:004945

Fig. 2. Phylogenomic relationship based on concatenated alignment of amino acid sequences between strains A1T, B1T and H1T and other related taxa of the family Comamonadaceae. The tree was reconstructed using the maximum-likelihood­ method based on concatenated alignment of amino acid sequences of 30 conserved marker genes coded in the genomes. Bootstrap values are shown at branch points.

Bligh–Dyer protocol [32, 33] followed by saponification the ratio of major fatty acid composition of strains A1T, H1T T with 0.5 M NaOH at 70 °C for 4 h. Extracts were separated and B1 , especially in ratio of C16 : 0, C16:1ω7c and C18 : 0. The into hydrocarbon and acid fractions using solid phase differences enable to discriminate the strains. The respira- extraction columns with an aminopropyl stationary phase tory quinone of strains A1T, H1T and B1T was ubiquinone, (Supelco). Fatty acids were analysed as methyl ester deriva- which was deduced from the coded genes of the respective tives. Double bond positions were determined by derivati- genomes (kegg module M00117). zation to dimethyl disulfide adducts following the methods of Shibamoto et al. [34]. Fatty acid derivatives and hydro- Phase contrast images were taken using a confocal micro- carbons were identified via GC-MS(Thermo­ Fisher Trace scope (LSM8000, Zeiss) equipped with an Orca-­Flach 4.0 GC and DSQ quadrupole mass spectrometer) and quanti- camera (Zeiss). Scanning electron microscopy images were fied using a coupled flame-­ionizing detector relative to an taken with JSM-7001 apparatus (jeol). For transmission internal standard [35]. Major fatty acid constituents were electron microscopy, the specimens negatively strained by consistent between strains, with strain A1T containing C EM stainer (Nisshin EM) for 5 min. Cells were observed 16 : 0 under a transmission electron microscope (JEM-­ARM200F, (3.3 %), C16:1ω7c (50.6 %), C18 : 0 (0.4 %) and C18:1ω7 (21.0 %), T jeol) operated at an accelerating voltage of 200 kV. These strain H1 containing C16 : 0 (27.8 %), C16:1ω7c (7.1 %), C18 : 0 T microscopic analyses revealed that the cell of strains A1T, H1T (5.9 %) and C18:1ω7c (22.6 %), and strain B1 containing and B1T features rod-­shaped, motile cells 1–3 µm long, with a C16 : 0 (12.9 %), C16:1ω7c (34.3 %), C18 : 0 (3.1 %) and C18:1ω7c (20.9 %). The fatty acid profiles were determined from a single polar (Fig. 3). single set of cultures with one replicate of each culture. Gram stain and catalase activity analyses revealed that all The major fatty acid constituents of strains A1T, H1T and T three strains were gram negative and catalase positive. The B1 (C16 : 0, C16:1ω7c and C18:1ω7c) are similar to those of the T T cytochromes were examined by sonicating whole cells, Malikia granosa P1 , Hydrogenophaga flava DSM 619 , and centrifuging at 10 000 g for 5 min, and performing a wave- Hydrogenophaga palleronii DSM 63T, but different from T length scan from 350 nm to 700 nm with a UV-Vis­ spec- those of Macromonas bipunctata DSM 12705 (Table 3). trometer (UV2600, Shimadzu). A difference spectrum was Interestingly, an unusual monosaturated nonadecanoic acid collected from air oxidized vs. dithionite reduced cell lysate. (omega 6) was found in all strains at low levels (A1T 0.9 %, The difference spectrum showed cytochrome peaks with H1T, 0.3 %, B1T 1.1 %) Further, a series of saturated and the maximum readings at 418–421 nm, and 550–553 nm. monounsaturated even-­chain linear hydrocarbons C16 to T T T T C28 was detected in all strains and strains A1 and H1 addi- Comparison of characteristics of the strains A1 , H1 and tionally contained squalene. There are some differences in B1T with the related genera in the family Comamonadaceae

4 Bird et al., Int. J. Syst. Evol. Microbiol. 2021;71:004945 no. Refseq accession GCF_000696225.1 GCF_000828895.1 GCF_000828915.1 GCF_002837135.1 GCF_002980595.1 GCF_002980625.1 GCF_001571145.1 GCF_001592285.1 GCF_001592305.1 GCF_000263795.2 GCF_000723405.1 GCF_001713375.1 GCF_001428625.1 GCF_001571225.1 1 2 1 1 93 82 43 53 34 115 162 103 124 110 No. of of No. sccafold 66.0 66.2 66.1 63.8 66.8 65.6 67.1 67.3 66.7 68.4 68.4 65.5 65.1 66.8 G+C (mol%) content (bp) 2 574 013 2 475 906 2 623 236 2 699 505 5 161 958 4 505 692 3 832 968 3 778 566 5 275 331 5 144 529 5 288 135 4 674 680 5 307 743 4 841 746 Total length length Total * 65 65 64 66 65 65 72 73 74 74 73 79 79 palleronii Hydrogenophaga Hydrogenophaga sp. * 64 65 64 66 65 64 73 74 76 73 73 89 Root209 80.5 (25.3) Hydrogenophaga sp. * 65 65 64 66 65 65 74 74 76 74 74 RAC07 86.5 (34.2) 80.7 (25.3) % for ANIb). % for Hydrogenophaga * 64 65 64 65 69 64 64 70 70 100 %). Bottom, ANIb values from JSpeciesWS and dDDH values derived from the from derived and dDDH values JSpeciesWS from ANIb values Bottom, %). 77.6 (21.5) 77.0 (21.7) 78.1 (22.6) intermedia Hydrogenophaga Hydrogenophaga sp. * % for dDDH and 96 % for 64 65 64 65 69 64 63 70 70 PBC off values (70 off 99.7 (99.2) 77.6 (21.5) 77.1 (21.8) 78.2 (22.6) Hydrogenophaga * 65 65 65 66 66 65 77 78 off values (70 off 77.1 (22.2) 77.2 (22.3) 79.5 (24.1) 79.2 (24.1) 79.4 (24.1) taeniospiralis Hydrogenophaga Hydrogenophaga * 65 65 65 67 66 66 94 80.2(24.3) 76.7 (21.9) 76.8 (21.8) 77.5 (21.9) 77.2 (22.0) 77.9 (22.7) psuedoflava Hydrogenophaga Hydrogenophaga * 65 65 64 66 66 65 flava 92.5(50.7) 80.4(24.5) 76.5 (21.4) 76.6 (21.4) 77.5 (21.8) 77.1 (21.8) 77.6 (22.5) Hydrogenophaga Hydrogenophaga * 65 66 65 71 90 spinosa Malikia Malikia 75.2(21.1) 74.9(21.5) 74.8 (21.1) 74.2 (20.7) 74.3 (20.8) 74.2 (20.9) 73.9 (20.6) 74.6 (21.0) * 65 66 65 71 Malikia Malikia granosa 75.6(21.2) 75.1(21.8) 89.8 (40.3) 75.1 (21.3) 74.6 (20.9) 74.3 (20.8) 74.3 (20.6) 74.4 (21.4) 74.8 (21.2) * 67 67 67 75.7(21.2) 75.9(21.8) 77.7 (22.4) 77.5 (22.4) 75.6 (21.3) 75.5 (20.6) 75.4 (20.6) 75.4 (20.8) 75.3 (20.7) 75.9 (21.2) bipunctata Macromonas * T 85 85 ­ genus cut- above are Bold letters based distance matrix calculator. B1 73.5(20.2) 73.6(20.4) 75.3 (20.5) 74.7 (20.9) 74.6 (20.8) 73.4 (19.9) 74.0 (20.8) 73.8 (20.5) 73.2 (20.3) 73.0 (19.9) 73.7 (20.6) * T 93 H1 73.5(19.8) 73.6(20.2) 85.0 (28.8) 75.3 (20.6) 74.6 (20.5) 74.4 (20.2) 73.2 (19.9) 73.6 (20.3) 73.5 (20.2) 73.2 (20.1) 73.1 (19.6) 73.7 (19.9) * T A1 92.0 (46.6) 85.3 (29.6) 75.1 (20.5) 74.4 (20.3) 74.4 (20.2) 73.2 (19.8) 73.3 (19.6) 73.5 (19.9) 73.5 (20.1) 73.4 (20.1) 73.0 (19.8) 72.8 (19.4) 73.5 (20.0) ­ species cut- above are Bold letters Genome Distance Calculator (in parentheses). to- ­ T T T A1 B1 H1 Macromonas Macromonas bipunctata Malikia granosa Hydrogenophaga flava Hydrogenophaga psuedoflava Hydrogenophaga taeniospiralis sp. Hydrogenophaga PBC Hydrogenophaga Hydrogenophaga intermedia sp. Hydrogenophaga RAC07 sp. Hydrogenophaga Root209 Hydrogenophaga Hydrogenophaga palleronii , Malikia and Hydrogenophaga Macromonas with genera strains the three for ANIb and dDDH results AAI, 1. Table the genome- ­ from AAI values Top, Genome- ­

5 Bird et al., Int. J. Syst. Evol. Microbiol. 2021;71:004945 7 − − − − + + − − + + 28 nt nt nt nt nt nd nd nd nd Slime 7.2–7.4 Oxygen Polar tuft Polar [11, 40]; 6, 40]; 6, [11, T 6 − + + + + + + + + + + 30 nt nt nt nt nd nd 7.2 <42 Polar Oxygen Pale yellow Pale Soil and water Soil and 5 − + + − − + + + + + + + − w 30 nt nd nd nd 7.2 <42 Mud Polar nitrate Oxygen DSM 619 flava Hydrogenophaga [6]; 5, T 4 − + − − − + + − + + − 35 nt nt nt nt nd <40 0-10 Polar nitrate 6.5–7.0 Oxygen Cream white Cream Activated sludge Activated 3 + − + + − + + − + − + w w w w 11 Polar 0–0.5 18–37 26–30 nitrate Oxygen 9.5–12.5 Opaque cream Opaque Serpentinized water P1 Malikia granosa this study and [19]); 4, (data from T , not tested or not available in the literature not tested or available nt , weak positive; w , negative; −, Positive; +, [6]. T 2 + + + + + − w w w 11 nt nt nt nt nt nt Polar 0–0.5 18–37 26–30 nitrate Oxygen 9.0–12.0 Opaque cream Serpentinized water from closely related type strains in the family Comamonadaceae in the family type strains related closely from T and B1 T 1 − + + + + − − + + − + + − w w 11 Polar 0–0.5 18–37 26–30 Oxygen (data from this study and [19]); 3, B1 this study and [19]); 3, (data from , H1 10.0–11.5 T thiosulfate T Opaque cream Opaque Serpentinized water Macromonas bipunctata DSM 12705 Macromonas 41]; 7, 40, [11, T ) −1 (data from this study and [19]); 2, H1 this study and [19]); 2, (data from T - ­ Lactate 2 dl Fermentation by glucose by Fermentation Characteristics Isolation source Isolation (°C) growth for Temperature (°C) growth for temperature Optimum Colony colour Colony pH range for growth for pH range Optimum pH for growth pH for Optimum (g l range tolerance NaCl Flagella Polyhydroxyalkanoate accumulation Polyhydroxyalkanoate Utilization of: Utilization H Thiosulfate Formate  Glucose Glycerol Acetate Butyrate Pyruvate Glutamate Cyclohexane Fumarate Propionate acceptors Electron DSM 63 palleronii Hydrogenophaga Differentiating characteristics of strains A1 of strains characteristics Differentiating 2. Table Strains: 1, A1 1, Strains:

6 Bird et al., Int. J. Syst. Evol. Microbiol. 2021;71:004945

Table 3. Major fatty acids (%) of strains A1T, H1T, B1T and their closely related type strains Strains: 1, A1T (data from this study and [19]); 2, H1T (data from this study and [19]); 3, B1T (data from this study and [19]); 4, Malikia granosa P1T [6]; 5, Hydrogenophaga flava DSM 619T [11, 40]; 6, Hydrogenophaga palleronii DSM 63T [11, 40, 41]; 7, Macromonas bipunctata DSM 12705T [6]. For unsaturated fatty acids, the position of the double bond is located by counting from methyl (ω) end of the carbon chain. nd, Not detected.

Fatty acid 1 2 3 4 5 6 7

Saturated:

C14 : 0 nd nd nd 1.4 2.8 0.4 2.4

C15 : 0 nd nd nd nd 0.4 nd 1.2

C16 : 0 3.3 27.8 12.9 14.7 19.4 31.6 5.3

C17 : 0 nd nd nd nd 1.3 17.8 0.8

C18 : 0 0.4 5.9 3.1 nd 1.5 0.6 nd

Unsaturated:

C15 : 1 ω6c nd nd nd nd nd 0.1 0.9

C16 : 1 ω5c nd nd nd 3.2 nd* nd 1.1

C16 : 1 ω7c 50.6 7.1 34.3 71.0 51.7* 25.0† 62.5

C17 : 1 ω6c nd nd nd nd nd nd 12.5

C17 : 1 ω8c nd nd nd nd nd nd 1.1

C18 : 1 ω7c 21.0 22.6 20.9 6.3 13.7* 19.5‡ 11.9

C19 : 1 ω6c 0.9 0.3 1.1 nd nd nd nd

Hydroxy:

C8 : 0 3-­OH nd nd nd nd 1.2 3.8 0.2

C10 : 0 3-­OH nd nd nd nd 2.9 nd nd

Cyclo propane:

C17:0 cyc nd nd nd nd <0.1 17.2 nd

*Summed feature comprised C16 : 1 or C18 : 1.

†Summed feature comprised C16 : 1 ω6c and/or C16 : 1 ω7c.

‡Summed feature comprised C18 : 1 ω6c and C18 : 1 ω7c. are summarized in Table 2. While genera in the family and 9.5, respectively. Genome size of strains A1T, H1T and Comamonadaceae harbour a remarkable phenotypic B1T was smaller than those of the closely related strains diversity, the strains A1T, H1T and B1T also share the of the family Comamonadaceae, while the G+C content phenotypic similarity with other genera in this family. was similar to those. Utilization of organic and inorganic For instance, genus Hydrogenophaga and strains A1T, H1T substrates, as well the ability to grow on alternative electron and B1T have a capability of autotrophic growth [11] and acceptors, was variable both between the strains and in the the genera Hydrogenophaga, Malika and strains A1T, H1T most closely related genera. Genomic comparison of the and B1T accumulate polyhydroxyalkanoate [6]. A notable carbon utilization genes among the three strains showed T T T physiological feature of strains A1 , H1 and B1 is the that only strain A1T encodes carbon monoxide dehydro- extremely high pH for the optimum growth (pH 11), which genase, benzoyl-­CoA-­oxygenase and phenylacetate-­CoA distinguishes it from closely related members, This high oxygenase, as described previously [19]. Regarding electron pH growth was confirmed in a continuous flow chemostat acceptor utilization, all three genomes encode genes for T T (BioFlo) at constant pH for strains A1 and B1 ; both strains oxygen respiration, while only, H1T and B1T encode genes showed growth at pH 11 under these conditions, and B1 for nitrate reductase, which agrees with the experimental continued to grow when the pH was shifted to pH 012. The results (the observed ability to grow anaerobically with optimum pH of 11 is the highest value for any prokaryote nitrate). reported so far [36]. Several species in the family Coma- monadaceae were reported as alkali tolerant or alkaliphilic, High sequence diversity of the strains A1T, H1T and B1T which include Ramlibacter alkalitolerans [37] and Mela- from members of family Comamonadaceae are represented minivora alkalimesophila [38] with the optimum pH 7.0 in the phylogenetic dendrograms based on 16S rRNA gene

7 Bird et al., Int. J. Syst. Evol. Microbiol. 2021;71:004945

Description of Serpentinimonas gen. nov. Serpentinimonas (​Ser.​pen.​ti.​ni.​mo′nas. N.L. neut. n. serpen- tinum a dark green mineral produced from reaction of olivine with water; L. fem n. monas a shape, a monad; N.L. fem. n. Serpentinimonas, a monad from a serpentinizing site.). Cells are Gram-stain-­ ­negative, rod shaped, motile cells 1–3 µm long, with a single polar flagellum. Organisms in this genus form small light-­coloured (opaque creamy) colonies on plates (<1 mm). Optimum growth occurs at 30 °C at pH 11. Preferred media is without NaCl. Cells are catalase posi- tive and sensitive to the , kanamycin (50 µg ml−1) and gentamicin (10 µg ml−1). Fatty acid profiles are simple,

containing primarily C16 : 0, C16:1ω7c, C18:0, C18:1ω7c and C19:1ω6c fatty acids as well as linear hydrocarbons. Respiratory quinone was ubiquinone. Phylogenetically, the genus is a member of the Comamonadaceae. Although we proposed the novel strains as the new genus candidatus ‘Serpentinomonas’ in our previous publication, the genus name is inappropriate as Latin name, based on the publication written by H.G. Trüper [39]. Therefore, here we propose the new genus name as Serpen- tinimonas. The type species is Serpentinimonas raichei.

Description of Serpentinimonas raichei sp. nov. Serpentinimonas raichei (rai′che.i. N.L. gen. n. raichei, named after R. Raiche, one of the owners of The Cedars nature reserve). In addition to the characteristics given above in the genus description, S. raichei has the characteristics described below. Growth occurs at 18–37 °C and pH 10.0–11.5 with optimal growth at 30 °C and pH 11.0. NaCl ranges from 0 to 0.5 g l−1. The DNA base composition of the type strain is 66.6 % G+C (determined from the genome). The strain grows autotrophi- cally with hydrogen gas and calcium carbonate and hetero- trophically on acetate, butyrate, lactate, pyruvate, ethanol, cyclohexane and fumarate under microaerophilic condition. Fig. 3. Microscopic observation of strain A1T. (a) Phase contrast The strain cannot utilize nitrate, sulphate, iron (III) hydroxide microscopy image of strain A1T grown on acetate and oxygen. The three or iron (II/III) oxide as electron acceptors. The strain cannot strains are visually indistinguishable. (b) SEM image of strain A1T on ferment glucose. Major fatty acids are C16:1ω7c and C18:1ω7c. carbon filter paper. Morphologies are indistinguishable for the three The respiratory quinone is ubiquinone. strains. (c) TEM image of strain A1T grown on acetate with oxygen. The type strain, A1T, (=NBRC 111848T=DSM 103917T), was isolated from a highly alkaline serpentinizing spring (Barnes Spring 1) in The Cedars located in north California, USA. sequences (Fig. 1) and concatenation of 30 conserved marker genes (Fig. 2). These relationships are also supported Description of Serpentinimonas by the low AAI values as shown in Table 1. Thus, strains barnesii sp. nov. A1T, H1T and B1T merit recognition as representative of a novel genus in the family Comamonadaceae. Further, due to Serpentinimonas barnesii (​bar.​ne′si.i. N.L. gen. n. barnesii, the low values of ANIb, dDDH and 16S rRNA gene identity, named after I. Barnes, geochemist and first describer of The strains A1T, H1T and B1T each represent distinct species in Cedars serpentinization site). this novel genus. Based on above findings, we propose that In addition to the characteristics given above in the genus strains A1T, H1T and B1T represent three novel species in a description, the type strain has the characteristics described new genus within the family Comamonadaceae. below. Growth occurs at 18–37 °C and pH 9.0–12.0 with

8 Bird et al., Int. J. Syst. Evol. Microbiol. 2021;71:004945

optimal growth at 30 °C and pH 11.0. NaCl ranges from 0 rhodoquinone producers to bacterial species with various quinone to 0.5 g l−1. The DNA G+C composition of the type strain is classes. J Gen Appl Microbiol 1995;41:99–117. 66.7 mol% (determined from the genome). The strain grows 3. Grabovich M, Gavrish E, Kuever J, Lysenko AM, Podkopaeva D, et al. Proposal of Giesbergeria voronezhensis gen. nov., sp. nov. and autotrophically with hydrogen gas and calcium carbonate G. kuznetsovii sp. nov. and reclassification of [Aquaspirillum] anulus, and heterotrophically on acetate, butyrate, lactate, pyruvate, [A.] sinuosum and [A.] giesbergeri as Giesbergeria anulus comb. ethanol and fumarate under microaerophilic conditions. The nov., G. sinuosa comb. nov. and G. giesbergeri comb. nov., and [Aquaspirillum] metamorphum and [A.] psychrophilum as Simpli- strain can ferment glucose. The strain is also able to utilize cispira metamorpha gen. nov., comb. nov. and S. psychrophila comb. glucose as an electron donor, and nitrate as an electron nov. Int J Syst Evol Microbiol 2006;56:569–576. acceptor. Major fatty acids are C16 : 0 and C18:1ω7c. The respira- 4. Dubinina GA, Grabovich MY. Isolation, cultivation, and characteris- tory quinone is ubiquinone. tics of Macromonas bipunctata. Microbiology 1984;53:610–617.

T T T 5. Heylen K, Lebbe L, de Vos P. Acidovorax caeni sp. nov., a denitrifying The type strain, H1 (=NBRC 111849 =DSM 103920 ), was species with genetically diverse isolates from activated sludge. Int isolated from a highly alkaline serpentinizing spring (Barnes J Syst Evol Microbiol 2008;58:73–77. Spring 5) in The Cedars located in north California, USA. 6. Spring S, Wagner M, Schumann P, Kämpfer P. Malikia granosa gen. nov., sp. nov., a novel polyhydroxyalkanoate- and polyphosphate-­ accumulating bacterium isolated from activated sludge, and reclassification of spinosa as Malikia spinosa comb. Description of Serpentinimonas nov. Int J Syst Evol Microbiol 2005;55:621–629. maccroryi sp. nov. 7. Yu XY, Li YF, Zheng JW, Li Y, Li L, et al. Comamonas zonglianii sp. Serpentinimonas maccroryi (​mac.​cro′ry.i. N.L. gen. n. nov., isolated from phenol-­contaminated soil. Int J Syst Evol Micro- biol 2011;61:255–258. maccroryi, named after D. McCrory, one of the owners of 8. Ding YP, Khan UK, Li MM, Xian WD, Liu L, et al. Calidifontimirobium The Cedars nature reserve). sediminis gen. nov., sp. nov., a new member of the family Como- In addition to the characteristics given above in the genus monadaceae. Int J Syst Evol Microbiol 2019;69:434–440. description, the type strain has the characteristics described 9. Ginige MP, Hugenholtz P, Daims H, Wagner M, Keller J, et al. Use of stable-­isotope probing, full-cycle­ rRNA analysis, and fluores- below. Growth occurs at 18–37 °C and pH 9.0–12.5 with cence in situ hybridization-­microautoradiography to study a meth- optimal growth at 30 °C and pH 11.0. The strain tolerates anol- fed denitrifying microbial community. Appl Environ Microbiol NaCl ranges from 0 to 0.5 g l−1. The DNA G+C composition 2004;70:588–596. of the type strain is 66.7 mol% (determined from the genome). 10. Finneran KT, Johnsen CV, Lovley DR. Rhodoferax ferrireducens The strain grows autotrophically on formate and hydrogen sp. nov., a psychrotolerant, facultatively anaerobic bacterium that oxidizes acetate with the reduction of Fe(III). Int J Syst Evol Microbiol gas but not on thiosulfate under microaerophilic conditions. 2003;53:669–673. The strain can use nitrate, but not thiosulfate as an electron 11. Willems A, Busse J, Goor M, Pot B, Falsen E, et al. Hydrogenophaga, acceptor. The strain can ferment glucose and grow hetero- a new genus of hydrogen-oxidizing­ bacteria that includes trophically on acetate, butyrate, lactate, pyruvate, ethanol, Hydrogenophaga flava comb. nov. (formerly Pseudomonas flava), glucose and fumarate. Major fatty acids are C , C ω7c and Hydrogenophaga palleronii (formerly Pseudomonas palleronii), 16 : 0 16:1 Hydrogenophaga pseudoflava (formerly Pseudomonas pseudoflava C18:1ω7c. The respiratory quinone is ubiquinone. and “Pseudomonas carboxydoflava”), and Hydrogenophaga taenio- T T T spiralis (formerly Pseudomonas taeniospiralis). Int J Syst Bacteriol The type strain, B1 (=NBRC 111850 =DSM 103919 ), was 1989;39:319 isolated from a highly alkaline serpentinizing spring (Barnes 12. Hesselmann RP, Werlen C, Hahn D, van der Meer JR, Zehnder AJ. Spring 1) in The Cedars located in north California, USA. Enrichment, phylogenetic analysis and detection of a bacterium that performs enhanced biological phosphate removal in activated sludge. Syst Appl Microbiol 1999;22:454–465. Funding information 13. Rouvière PE, Chen MW. Isolation of Brachymonas petroleovorans This work was funded by the NSF-­EAR (Grant Nos.1424646 and CHX, a novel cyclohexane-­degrading beta-­proteobacterium. FEMS 1424711). This work was partly supported by the JSPS Grand-in-­ ­Aid Microbiol Lett 2003;227:101–106. for Science Research (Nos. 20H04620 and 18H02501). 14. Morrill PL, Kuenen JG, Johnson OJ, Suzuki S, Rietze A, et al. Acknowledgements Geochemistry and geobiology of a present-day­ serpentiniza- We greatly appreciate Roger Raiche and David McCrory for providing tion site in California: The Cedars. Geochim Cosmochim Acta their private land for our research. We appreciate Orion Johnson, Greg 2013;109:222–240. Wanger and Orianna Bretschger for the field support and Koji Mori for 15. Szponar N, Brazelton WJ, Schrenk MO, Bower DM, Steele A, et al. constructive discussions. The advice on name giving by Latinist Dr. A.H. Geochemistry of a continental site of serpentinization, the Table- van der Laan (University of Groningen, NL) is gratefully acknowledged lands Ophiolite, Gros Morne National Park: A Mars analogue. Icarus 2013;224:286–296. Conflicts of interest The authors declare that there are no conflicts of interest. 16. Marques JM, Carreira PM, Carvalho MR, Matias MJ, Goff FE, et al. 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